Exploration of Bacterial Re-Growth as In Vitro Phenomenon Affecting Methods for Analysis of the Antimicrobial Activity of Chimeric Bacteriophage Endolysins.

Ursula Kaspar,Nina Schleimer,Karsten Becker,Evgeny A Idelevich,Sonja Molinaro

doi:10.3390/microorganisms10020445

Abstract

Drug alternatives to combat methicillin-resistant Staphylococcus aureus (MRSA) in human and animal healthcare are urgently needed. Recently, the recombinant bacteriophage endolysins, PRF-119 and its successor substance HY-133, have proven to be highly active against various S. aureus clonal lineages and to exhibit a very rapid bactericidal effect when standard methods for susceptibility testing are applied. Along with subsequent growth curve experiments, a re-growth phenomenon was observed in vitro necessitating its clarification for the assessment of the agent’s stability and activity as well as for methodological aspects of endolysin testing in general. Distinct in vitro parameters were comparatively examined applying also scanning electron microscopy, fluorescence assays and SDS-PAGE analysis. The shape and material of the culture vessels as well as the shaking conditions were identified as factors influencing the in vitro stability and activity of HY-133. The highest function maintenance was observed in plain centrifuge tubes. Based on this, the conditions and parameters of assays for testing the antimicrobial activities of phage endolysins were determined and adjusted. In particular, shear forces should be kept to a minimum. Our results form the basis for both future test standardization and re-growth-independent experiments as prerequisites for exact determination of the antimicrobial activities of engineered endolysins.

Highlights

Introduction iationsMethicillin-resistant Staphylococcus aureus (MRSA) is a major cause of community- and healthcare-associated human and animal infections worldwide [1]
Median Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of each 0.5 μg/mL of HY-133 were determined for S. aureus ATCC 29213
MICs of HY-133 were applied in time–kill experiments

Summary

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of community- and healthcare-associated human and animal infections worldwide [1]. As it is a serious threat for individual patients and additional socioeconomic burden for healthcare systems, infection control measures including de-colonization strategies may help to prevent infections caused by healthcare-, community- and livestock-associated MRSA [2,3,4,5]. In particular, bacteriophage-derived lysins have recently been considered as an alternative therapeutic strategy to combat the escalating problem of resistance in human and animal healthcare [10]. HY-133 (HYPharm GmbH, Bernried, Germany) is a chimeric bacteriophage endolysin, which may open novel strategies for nasal de-colonization of patients carrying S.

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